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This dissertation, Nuclear Transcription Factors and Hypoxia-inducible Genes in Chronic Liver Hypoxia by Yue-huen, Thomas, Lau, 劉汝這, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Nuclear transcription factors and hypoxia- inducible genes in chronic liver hypoxia submitted by Thomas Yue- Huen Lau for the degree of Doctor of Philosophy at The University of Hong Kong in June 2005 Cellular metabolism of mammals depends on a constant supply of O . Severe chronic hypoxia induces apoptosis with functional disturbance of body organs. Studying the molecular adaptive mechanisms in chronic liver hypoxia provides a foundation in managing treatment of tumoral and non-tumoral conditions in which prolonged O deprivation could be the underlying cause leading to liver failure. To investigate hepatic adaptation in chronic hypoxia, adult male Sprague-Dawley rats inspiring 10% O for 28 days in an isobaric hypoxic chamber were used. Blood and liver samples 2 were collected at four time points (day 7, day 14, day 21 and day 28). Body weights, histology, serum assays for hematocrit, alanine aminotransferase (ALT) and total 8-isoprostane were used to assess the status of hematopoietic response, liver function and oxidative stress. EMSA, RT- PCR, Western Blotting and immunohistochemistry (IHC) were used to study the molecular events in hepatic adaptation to hypoxia. Expression of nitrotyrosine, hypoxia-inducible factor-1α (HIF-1α), nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1) and hypoxia-inducible genes including VEGF, iNOS, ET-1, GLUT-1 and GLUT-3 were assessed. The hypoxia- inducible genes contain the hypoxia-response element (HRE) and transcriptionally regulated by HIF-1. eNOS and GLUT-2 (non-HRE genes) were also assessed. iNOS and eNOS transcription are regulated by NF-κB and AP-1 respectively. 1 A difference in body weights were recorded between the chronically hypoxic (CH) and the respective normoxic control (C) groups of rats at all time points. From day 14 onward, significant increase in the hematocrit level confirmed persistent systemic response of the CH rats to chronic hypoxia. At all time points, histology showed normal hepatic morphology in the CH and C groups. The serum ALT, total 8-isoprostane and nitrotyrosine levels were not significantly elevated in the CH groups indicating the absence of oxidative stress. EMSA showed significant increase in the DNA binding activities of HIF-1α, NF-κB and AP-1 at all time points in the CH groups and IHC identified pronounced expression of HIF-1α at the sinusoidal cells and the hepatocytes. RT-PCR showed significant increase in VEGF, iNOS, eNOS and ET-1 mRNAs expression in the CH groups. IHC identified the main sources of VEGF and iNOS from the hepatocytes. eNOS and ET-1 were expressed in the sinusoidal and centrilobular endothelial cells respectively. The protein expressions of these genes were confirmed by Western Blotting that showed similar trends as their mRNAs expression. IHC identified GLUT-1 expression in the centrilobular endothelial cells and GLUTs 1, 2 and 3 in the hepatocytes. Western Blotting confirmed significant expressions of the GLUTs in the CH groups giving evidence of facilitated glucose uptake and transportation. In chronic liver hypoxia, HIF-1α is the key transcription factor that mediates VEGF, ET- 1 and iNOS gene activities in sustaining cellular integrity. NF-κB and AP-1 enhance the cytoprotective properties of nitric oxide (NO) via transactivation of iNOS and eNOS respectively. Hence, the hepatic adaptive re

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